Cholesterol delivered to cells via uptake of low density lipoproteins traverses the endocytic pathway to sites of utilization and regulation of cellular cholesterol homeostasis. The endocytic pathway is complex consisting of various organelles including early and late endosomes and lysosomes, as well as traffic flow that may be bidirectional. Our studies have concentrated on the intracellular pathway of cholesterol transport in normal and mutant cells. Endocytic cholesterol transport is interrupted in the human genetic mutation Niemann Pick Type C disease, resulting in pathological accumulation of free cholesterol intracellularly. The defective gene responsible for NPC disease predicts a protein (NPC1) that has homology to other known proteins involved in cholesterol metabolism. Structural immuno-cytochemical studies combined with biochemical and genetic assays resulted in the elucidation of the role played by NPC1 protein in cholesterol sensitive translocation of cellular lipids. The NPC1 protein resides in a late endosomal organelle that interacts with cholesterol laden lysosomes to effect the movement of free cholesterol to plasma membrane and the endoplasmic reticulum, the site of cholesterol esterification and stimulation of homeostatic responses. In addition the NPC1 containing endocytic vesicle plays a role in sorting of glycolipids for further transport to cholesterol laden lysosomes or other cellular locations. Studies in living cells, using a green fluorescent and NPC1 chimeric protein, show that NPC1 mediated trafficking of lipids in cells occurs by means of membraneous tubules that originate and flow from components of the endocytic pathway. Further studies on site directed mutagenesis of the NPC1 protein have targeted domains of the protein responsible for its cholesterol transport function. Our studies are designed to obtain knowledge of the controlling factors responsible for membrane traffic of NPC1 protein in normal cells and elucidate the defect in these functions in mutant NPC1 cells. - Cholesterol Niemann Pick Type c Immunocytochemistry Golgi Lysosomes Cell Biology